Reading frame maintenance by the ribosome during stalling

项目摘要/摘要 在生命的所有领域，将遗传信息解码成多肽是由核糖体完成的， 它一次读取信使RNA(MRNA)三个核苷酸。在仔细挑选了 与这个三联体密码子匹配的氨酰tRNA，核糖体必须准确地移动到阅读下一个密码子。 精确的易位并非易事，因为mrna、trna和 必须发生的核糖体亚基。如果不这样做，会导致所谓的帧移位错误，即 不利于蛋白质平衡，因为它们导致错误的蛋白质产物与编码的 一个。值得注意的是，我们对阅读框维护的大部分了解都来自于对程序化阅读框架的研究 或者是“故意”的移帧。这些研究揭示了该基因的序列和结构特征。 它与核糖体元件、翻译因子和tRNA的相互作用促成了这些事件。 尽管这些元件中有许多是每个信使核糖核酸所特有的，但几乎所有的移码事件都依赖于核糖体。 在拖延时间。细胞对失速的反应几乎完全是在质量控制和核糖体的背景下 救援队。特别是，当它们引起核糖体碰撞时，它们被泛素连接酶识别。在……里面 原理上，核糖体的碰撞也可以提供移码所需的结构障碍；事实上，我们 最近的研究表明，碰撞可以导致有效的1移码，这表明细胞一定是进化出来的 翻译停顿期间维持阅读框架的因素。因为核糖体似乎经常在 在压力下，这些机制很可能成为细胞生存和恢复的关键。这项建议 集中在一种最近发现的机制上，它涉及高度保守的多蛋白质桥接 系数(Mbf1)。我们的初步研究表明，该因子可以防止碰撞介导的1帧移位。 这与Mbf1结合的核糖体的初步cyoEM结构一起构成了我们主要的 假设Mbf1识别碰撞的核糖体以防止它们改变阅读框架 领头羊。我们将通过三个目标来检验这一假设。在第一个问题中，我们将评估如何改变 核糖体密度和mRNA序列和结构特征调节Mbf1的功能，以努力 建立核糖体碰撞和移码之间的关系。在第二个目的中，使用修改后的 核糖体分析方法评估移码转录，我们将剖析Mbf1的作用 在防止随机碰撞以及在压力下经历的帧移位方面。在……里面 第三个目标，Mbf1对停滞的核糖体的募集机制将用一组 生物化学和生物物理方法。我们最感兴趣的是调查这一因素如何改变 翻译机的功能和结构。总体而言，我们的跨学科方法建立和 对已有的专业知识和资源进行扩展，我们计划使用这些专业知识和资源来揭示有关 保守因子如何被招募到核糖体以调节其在应激状态下的功能。

PROJECT SUMMARY/ABSTRACT In all domains of life, decoding of the genetic information into peptides is accomplished by the ribosome, which reads the messenger RNA (mRNA) three nucleotides at a time. Following careful selection of the aminoacyl-tRNA that matches this triplet codon, the ribosome must precisely move to reading the next codon. Precise translocation is not an easy task given the multiple coordinated movements of the mRNA, tRNA and the ribosomal subunits that must occur. Failure to do so results in so-called frameshifting errors, which are detrimental to proteostasis as they result in errant protein products that bear no resemblance to the encoded ones. Notably, much of what we know about reading-frame maintenance comes from studies on programmed or “intentional” frameshifting. These studies revealed that sequence and structural features of the mRNA and its interaction with elements of the ribosome, translation factors and the tRNA contribute to these events. Although many of these elements are unique to each mRNA, almost all frameshifting events rely on ribosome stalling. Cellular response to stalls has been almost exclusively in the context of quality control and ribosome rescue. In particular, stalls are recognized by ubiquitin ligases when they cause ribosome collisions. In principle, colliding ribosomes can also provide structural impediments required for frameshifting; indeed, we recently showed that collisions can lead to efficient +1 frameshifting, suggesting that cells must have evolved factors to maintain reading frame during translation stalls. As ribosomes appear to stall frequently under stress, these mechanisms are more than likely to become critical for cell survival and recovery. This proposal is focused on one recently identified mechanism that involves the highly conserved multi-protein bridging factor (Mbf1). Our preliminary studies suggest that the factor prevents collision-mediated +1 frameshifting. This, together with a preliminary cyoEM structure of a Mbf1-bound ribosome, forms the basis of our major hypothesis that Mbf1 recognizes collided ribosomes to prevent them from altering the reading frame of the leading one. We will test this hypothesis through three aims. In the first one, we will assess how altering ribosome density and mRNA-sequence and -structural features modulate the function of Mbf1 in an effort to establish a relationship between ribosome collisions and frameshifting. In the second aim, using modified ribosome-profiling approaches to assess frameshifting transcriptome-wide, we will dissect the role of Mbf1 in preventing frameshifting occurring at stochastic collisions as well as those experienced under stress. In the third aim, the mechanism of Mbf1 recruitment to stalled ribosomes will be studied using a battery of biochemical and biophysical approaches. We are most interested in investigating how the factor alters the function and the structure of the translation machinery. Collectively, our interdisciplinary approach builds and expands on established expertise and resources, which we plan to use to uncover important details about how conserved factors are recruited to ribosomes to modulate their function under stress.